UV-HYPERMUTABILITY OF XERODERMA-PIGMENTOSUM CELLS DEMONSTRATED WITH ADNA-BASED MUTATION SYSTEM

We have developed a DNA-based system, to detect mutations at restricti on sites without any selection in culture. DNA is exhaustively digeste d with a restriction enzyme. Primers Ranking a chosen site for this en zyme are used in the polymerase chain reaction (PCR). Only DNA molecul es mutated at the chosen site are resistant to digestion and can serve as templates for the PCR. We have initially used this system to demon strate the generation of mutations by ethyl methanesulphonate (EMS) at a TaqI site in the aprt gene of Chinese hamster cells, and by u.v.-C irradiation at a TaqI site in the hprt gene of human fibroblasts, In r epair-deficient xeroderma pigmentosum (XP) cells the u.v.-induced muta nt frequency was greatly enhanced. We have been able to detect and ana lyse mutations in XP cells at TaqI sites in three different genes, hpr t, p53 and c-Ha-ras1. Both u.v.-C and u.v.-B irradiation have been use d as mutagenic agents with both lymphoblastoid and fibroblast cells fr om XP patients from complementation group G. The mutant DNA molecules have been sequenced. Following u.v.-C-irradiation, the majority of mut ations analysed were GC-->AT transitions, but several double and tande m mutations were also found.